Induction and Characterization of a Microsomal Flavonoid 3′‐Hydroxylase from Parsley Cell Cultures

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Abstract
A microsomal preparation from irradiated parsley cell cultures catalyzes the NADPH and dioxygen-dependent hydroxylation of (S)-naringenin [(S)-5, 7, 4''-trihydroxyflavanone] to eriodictyol (5, 7, 3'', 4''-tetrahydroxyflavanone). Dihydrokaempferol, kaempferol and apigenin were also substrates for the 3''-hydroxylase reaction. In contrast, prunin (naringenin 7-O-.beta.-glucoside) was not converted by the enzyme. The microsomal preparation, which also contains cinnamate 4-hydroxylase, did not catalyze hydroxylation of 4-coumaric acid to caffeic acid. 3''-Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+. These properties suggest that the enzyme is a cytochrome P-450-dependent flavonoid 3''-monooxygenase. Pronounced differences in the inhibition of flavonoid 3''-hydroxylase and cinnamate 4-hydroxylase were found with EDTA, KCN and N-ethylmaleimide. Irradiation of the cell cultures led to an increase of flavonoid 3''-hydroxylase activity, with a maximum at about 23 h after onset of irradiation, and a subsequent decrease. This is similar to light-induction of phenylalanine ammonia-lyase and cinnamate 4-hydroxylase. In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f. sp. glycinea did not induce flavonoid 3''-hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and NADPH-cytochrome reductase. Flavonoid 3''-hydroxylase and cinnamate 4-hydroxylase are evidently 2 different microsomal monooxygenases.